Gene expression regulatory DNA

ABSTRACT

The present invention provides a gene expression regulatory DNA and a process for preparing a protein using the same. 
     A DNA derived from the isocitrate lyase (ICL) gene of a coryneform bacterium regulates expression of a structural gene encoding a protein when incorporated into a vector DNA together with said structural gene and introduced into a host coryneform bacterium, and a useful protein can be efficiently produced using the DNA.

BACKGROUND OF THE INVENTION

The present invention relates to a novel DNA which is obtained from acoryneform bacterium and which regulates expression of a structural geneand to a process for efficiently producing a useful protein using theDNA. "Coryneform bacteria" hereinafter refers to microorganismsbelonging to the genus Corynebacterium, Brevibacterium orMicrobacterium.

Development of recombinant DNA technology has enabled utilization ofvarious microorganisms for the production of useful polypeptides whichare products of heterologous organisms. However, when a genetic productof a foreign gene is toxic to a host, expression of the foreign genefrom the beginning of growth of the host causes death or growthinhibition of the host. Therefore, it is difficult to produce thegenetic product in large quantities. To overcome such problem, it hasbeen necessary to employ an expression system for inducing theexpression of the foreign gene after the growth phase of a hostmicroorganism. In E. coli which is most frequently used as a host, theexpression systems for genes utilizing promoters working in response toa specific compound or under specific physical conditions have beenestablished [Goeddel, D. , et al., Proc. Natl. Acad. Sci. U.S.A., 76,106 (1979) , Edman, J. C., et al., Nature, 291, 503 (1981), Shimatake,H., et al., Nature, 292, 128 (1981)]. By use of these systems, a varietyof useful proteins have been produced.

On the other hand, recombinant DNA technology is also applicable tocoryneform bacteria which are used for production of various aminoacids, purine nucleotides, and the like by fermentation. For example, apromoter for coryneform bacteria for structurally expressing a reportergene has been obtained using a vector for detecting a promoterfunctioning in such bacteria (EP-A-271838). However, there are noreports of the successful development of a promoter for regulatingexpression in coryneform bacteria. On the other hand, a method forartificially regulating expression of a foreign gene in a coryneformbacterium using the aforesaid promoter for E. coli capable of inducingexpression of a foreign gene in E. coli to induce the expression of thechloramphenicol acetyltransferase gene in coryneform bacterium has beenreported (EP-B-215388). However, this method for expression is notsufficiently potent and the yield of the genetic product accumulated issmall as compared with the yield obtained by using E. coli as the host.

Accordingly, in order to efficiently produce useful genetic products incoryneform bacteria, it is necessary to develop a DNA which functions insuch host bacteria and which enables artificial regulation of expressionof a structural gene.

SUMMARY OF THE INVENTION

The present invention provides a DNA and method by which expression isregulated in coryneform bacteria in response to environmental conditionssuch as the composition of the medium. More specifically, it has beenfound that the expression of isocitrate lyase (hereinafter referred toas ICL) gene of such bacteria is repressed when the carbon sources in amedium are sugars such as glucose, sucrose and maltose. When the carbonsources in the medium are non-sugars such as acetic acid, lactic acidand ethanol, or in the absence of sugars, expression is induced and thelevel of the expression is extremely high. A DNA fragment encoding theICL gene has been cloned and the nucleotide sequence of a DNA whichregulates the expression of the gene has been determined. It has thusbeen found that the DNA is novel and a desired gene can be efficientlyexpressed in coryneform bacteria, using this DNA.

In the amino acid fermentation by coryneform bacteria by knownprocesses, the culture containing microbial cells is usually discardedafter sugars in the medium is consumed and fermentation is completed.Introduction of the DNA of the present invention into a coryneformbacterium enables use of the culture for producing a protein under theconditions where no sugars are present. Therefore, by the presentinvention, the culture of a coryneform bacterium used for amino acidfermentation can be reutilized for the production of a protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a restriction map of a cloned Hind III-cleaved DNA fragment of6.0 kb carrying the ICL gene.

FIG. 2 shows the steps for preparing pKT10.

FIG. 3 is a restriction map of a cloned Hind III-cleaved DNA fragment of6.0 kb carrying the ICL gene in subcloning.

FIG. 4 shows the steps for preparing pCGKK27-3.

FIG. 5 shows the steps for preparing pKT22.

FIG. 6 shows the steps for preparing pKT23.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, a DNA derived from the ICL gene of acoryneform bacterium is incorporated into a vector DNA together with astructural gene encoding a protein and then introduced into a coryneformhost microorganism. The DNA (hereinafter referred to as ICL promoter)regulates the expression of the structural gene in the host. Moreparticularly, the expression of the structural gene is repressed whencarbon sources in the medium are sugars, and the expression is inducedwhen carbon sources in the medium are non-sugars or no sugars arepresent in the medium.

A DNA carrying the ICL promoter and ICL structural gene may be isolatedfrom any coryneform bacteria, but the following strains are preferablyused.

Corynebacterium glutamicum ATCC 13032

Corynebacterium acetoacidophilum ATCC 13870

Corynebacterium acetoglutamicum ATCC 15806

Corynebacterium callunae ATCC 15991

Corynebacterium herculis ATCC 13868

Corynebacterium melassecola ATCC 17965

Corynebacterium lilium ATCC 15990

Brevibacterium immariophilum ATCC 14068

Brevibacterium saccharolyticum ATCC 14066

Brevibacterium thiogenitalis ATCC 19240

Brevibacterium divaricatum ATCC 14020

Brevibacterium flavum ATCC 14067

Brevibacterium lactofermentum ATCC 13869

Brevibacterium roseum ATCC 13825

Microbacterium ammoniaphilum ATCC 15354

Extraction of a chromosomal DNA from coryneform bacteria is carried outby known procedure, for example, the method described in JapanesePublished Unexamined Patent Application No. 126789/83. The DNA fragmentcontaining the ICL gene region can be isolated from this chromosomal DNAby incorporating the chromosomal DNA treated with an appropriaterestriction enzyme into a plasmid or a phage DNA treated with the samerestriction enzyme or a restriction enzyme which causes the samecohesive end, transforming a microorganism with the resultantrecombinant DNA, and then isolating a clone carrying the recombinant DNAcontaining the desired DNA fragment.

For example, when E. coli is used as a host for transformation and aplasmid is used as a vector, the clone carrying the DNA fragmentcontaining the ICL gene on the plasmid vector can be isolated bydetecting a transformant carrying the DNA fragment which hybridizes to asynthetic DNA encoding a part of the amino acid sequence of ICL usingcolony hybridization [Hanahan, D. et al., Gene, 10, 63 (1980)]. Thesynthetic DNA used as a probe may be obtained, for example, by isolatingICL, determining the amino acid sequence at the N-terminus thereof, andchemically synthesizing a polynucleotide corresponding to the sequencein a conventional manner [M. H. Caruther et al., Chemical Synthesis ofGene Fragments, a Laboratory Manual, Verlag, Chemie (1982)]. A series offundamental operations required for the cloning in E. coli are known anddescribed in detail in Molecular Cloning (1982), Cold Spring HarborLaboratory.

By introducing the cloned fragment obtained by the above method intocoryneform bacteria, it can be confirmed that the cloned fragmentcarries the ICL promoter and the structural gene encoding ICL. For thispurpose, the E. coli plasmid carrying the cloned fragment is ligated toa vector capable of autonomous replication in coryneform bacteria, orthe cloned fragment is inserted in a vector for coryneform bacteria, toprepare a recombinant plasmid. Such recombinant DNA can be prepared byin vitro recombination, followed by transformation of a coryneformbacteria strain and selection of a transformant carrying a plasmidhaving the desired structure. Any vector may be used as the vector forcoryneform bacteria so long as it is capable of autonomous replicationin such bacteria. Examples of suitable vectors are pCG1 (JapanesePublished Unexamined Patent Application No. 134500/82), pCG2 (JapanesePublished Unexamined Patent Application No. 35197/83), pCG4 (JapanesePublished Unexamined Patent Application No. 183799/82), pAM330 (JapanesePublished Unexamined Patent Application No. 67699/83), pAG1, pAG3,pAG14, pAG50 (Japanese Published Unexamined Patent Application No.166890/87) and plasmids derived therefrom. In order to prepare plasmidsfrom coryneform bacteria, a known method, for example, the methoddescribed in Japanese Published Unexamined Patent Application No.134500/82 may be used. Transformation of coryneform bacteria is carriedout by a method using protoplasts (e.g., Japanese Published UnexaminedPatent Application No. 186492/82) or electroporation [Appl. Microbiol.Biotechnol., 30, 283 (1989)].

The thus prepared recombinant DNA is used to transform an ICLactivity-deficient mutant coryneform bacteria strain. If thetransformants obtained have acquired the ability of ICL synthesis, itindicates that the ICL gene is present on the cloned fragment.Alternatively, a wild strain having ICL activity may be transformed withthe recombinant plasmid. In such case, the presence of the ICL gene canbe confirmed by an increase in ICL activity as compared with the hostwhen cultured in a medium containing non-sugars such as acetic acid,lactic acid and ethanol as carbon sources, or in a medium where no sugaris present. From positive transformants, plasmids are extracted. Bydigestion of the plasmids with restriction enzymes followed by agarosegel electrophoresis, a DNA fragment containing the inserted ICL gene canbe isolated.

The ICL gene-carrying DNA fragment is subcloned and deletion plasmidsincluding various degraded small fragments are examined for ICLactivity-conferring ability or ICL activity-enhancing ability, wherebythe presence of ICL gene can be further specified. The nucleotidesequence of the DNA fragment carrying the ICL gene can be determined bythe dideoxynucleotide synthesis chain termination method [J. Mol. Biol.,94, 441 (1975)], the Maxam-Gilbert method [Proc. Natl. Acad. Sci., 74,560 (1977)], or by similar known methods. An open reading frame can bepresumed by finding the nucleotide sequence encoding the N-terminalamino acid sequence of ICL on the DNA nucleotide sequence. Based on thepresence of the open reading frame, it is assumed that the region havingthe ICL promoter activity is present upstream of the open reading frame.

By such analysis, in the case of, for example, Corynebacteriumglutamicum ATCC 13032 described in the examples, the ICL promoteractivity of the strain can be specified to be attributed to the sequenceat positions 1 through 513 on the DNA nucleotide sequence shown by Seq.ID NO:3. The ICL promoter activity of the present invention is notlimited to this DNA sequence. The DNA sequence may be partly deleted ormodified as long as the promoter activity is not damaged.

An expression vector can be obtained by inserting a DNA which containsthe DNA fragment having the ICL promoter activity, and downstream fromthe fragment, a structural gene and the terminator for terminatingtranscription into the above-mentioned plasmid capable of autonomousreplication in coryneform bacteria. As the terminator, it is preferredto use the terminator of the ICL gene, but terminators from other genesof coryneform bacteria or ρ-independent terminator derived from E. colior B. subtilis genes [Ann. Rev. Genet., 13, 319 (1979)] may also beused. Examples of appropriate structural genes are those of enzymes suchas β-galactosidase, chloramphenicol acetyltransferase and ICL, andphysiologically active proteins such as insulin, growth hormone, α-, β-or γ-interferon and granulocyte colony stimulating factor (G-CSF).

A host is transformed with the expression vector described above and thetransformants are cultured to express the desired genetic product. Asthe host, it is preferred to use the aforesaid coryneform bacteria, butother coryneform bacteria may also be used.

By culturing the transformants in a medium containing, as carbonsources, non-sugars such as acetic acid and lactic acid, and furthercontaining nitrogen sources, inorganic materials, vitamins, etc., thedesired genetic products are accumulated in the medium. Alternatively,the transformants are initially grown in a medium containing, as carbonsources, sugars such as glucose, sucrose and maltose, and after thesugars are consumed, the non-sugar carbon sources described above areadded to the medium or the medium is replaced with the one containing nosugar, followed by further culturing to obtain the desired geneticproducts.

Culturing is carried out under aerobic conditions with aeration orstirring. In general, it is preferred to keep the pH of the mediumaround neutrality during the culturing. Conditions such as culturingtemperature and time are adjusted to provide maximum proliferation ofthe host microorganism and the maximum production of the geneticproducts by the transformants. Generally, it is suitable to carry outculturing at a temperature of 15° to 40° C. for 4 to 72 hours.

The genetic products accumulated in the culture are extracted bydisrupting the microbial cells in conventional manner, for example, bymechanical disruption or by a method using a bacteriolytic enzyme.Isolation and purification of the desired genetic products from theextract can be carried out by combinations of methods conventionallyused for the purification of proteins, for example, precipitation usinga precipitating agent, dialysis, electrophoresis, chromatography usingion exchange resin or the like, gel filtration and a method using anantibody column.

Certain embodiments of the present invention are illustrated in thefollowing representative examples.

EXAMPLE 1 Mode of Expression of ICL Gene of Coryneform bacteria

In this example, the following coryneform bacteria were used:Corynebacterium glutamicum ATCC 13032, Corynebacterium acetoacidophilumATCC 13870, Corynebacterium callunae ATCC 15991, Corynebacteriumherculis ATCC 13868, Corynebacterium lilium ATCC 15990, Brevibacteriumimmariophilum ATCC 14068, Brevibacterium divaricatum ATCC 14020,Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum ATCC13655 and Microbacterium ammoniaphilum ATCC 15354. One loopful of eachof the strains was inoculated into NB medium, which is a mediumcontaining 20 g of bouillon powder, 5 g of yeast extract and 10 g ofglucose in 1 liter of water and adjusted to pH 7.2, followed by shakeculture at 30° C. for 16 hours for proliferation. The resulting seedcultures (0.8 ml) were inoculated into both semi-synthetic MAYE mediumcontaining acetic acid as the carbon source [medium containing 20 g ofammonium acetate, 10 g of (NH₄)₂ SO₄, 3 g of urea, 1 g of yeast extract,1 g of KH₂ PO₄, 0.4 g of MgSO₄.7H₂ O, 2 mg of FeSO₄.7H₂ O, 2 mg ofMnSO₄.4H₂ O, 60 μg of biotin, 2 mg of thiamine hydrochloride and 50 mgof NaCl in 1 liter of water and adjusted to pH 7.2], and MSYE mediumcontaining sucrose as the carbon source [medium containing 20 g ofsucrose, 10 g of (NH₄)₂ SO₄, 3 g of urea, 1 g of yeast extract, 1 g ofKH₂ PO₄, 0.4 g of MgSO₄.7H₂ O, 2 mg of FeSO₄.7H₂ O, 2 mg of MnSO₄.4H₂ O,60 μg of biotin, 2 mg of thiamine hydrochloride and 20 mg of NaCl in 1liter of water and adjusted to pH 7.2], followed by incubation at 30° C.for 16 hours.

The cells were collected, washed twice with 100 mM phosphate buffer (pH7.0), and then suspended in 5 ml of the same buffer. Under ice cooling,the cell suspension was subjected to cell disruption for 15 minutesusing a ultrasonic homogenizer (manufactured by TOMY Co., pencil typesonic). The homogenate was centrifuged at 4° C. for 10 minutes (14000×g)and the supernatant was recovered as the cell extract.

The ICL activity of the cell extract was determined by the method forquantitatively determining glyoxylic acid formed using isocitric acid asthe substrate [J. Biochem., 64, 355 (1968)]. That is, to 2.0 ml of areaction mixture [0.14M Tris-HCl (pH 7.5), 20 mM MgSO₄.7H₂ O and 20 mMglutathione] previously warmed to 30° C., were added the cell extract inan amount corresponding to 30 μg when calculated as protein and 20 μl of0.4M isocitric acid solution. The reaction was carried out at 30° C. for10 minutes and terminated by adding 1 ml of 0.5M oxalate solution to thereaction mixture. Following addition of 0.5 ml of 1% phenylhydrazinesolution, the reaction mixture was heated at 70° C. for 10 minutes,followed by cooling in ice water for 5 minutes. Then, 2 ml of conc.hydrochloric acid and 0.5 ml of 0.5% potassium ferricyanide solutionwere added to develop a color and the absorbance was measured at 520 nmusing a Hitachi Colorimeter (Model 100-20). The specific enzymaticactivity per mg of protein was calculated and indicated as unit (U)/mgprotein, one unit being defined as that enzymatic activity whichcatalyzes the formation of 1 μmol of glyoxylic acid in one minute. Theresults are shown in Table 1. The amount of protein was determined usinga Protein Assay Kit (manufactured by BIO-RAD Co.).

In all the strains, the ICL activity was only slightly detected or notdetected in the cells cultured in MSYE medium, whereas the ICL activityat high levels was noted in the cells cultured in MAYE medium. When thestrains were cultured in media containing, as carbon sources, sugarssuch as glucose, maltose and gluconic acid, the ICL activity was veryslight or not detected as in the case of the sucrose-containing medium.When the strains were cultured in media containing, as carbon sources,non-sugars such as lactic acid, ethanol and pyruvic acid, the ICLactivity at the same level as in the case of the acetic acid-containingmedium was detected.

The foregoing results confirmed that the expression of the ICL gene ofall the coryneform bacteria used in the test was repressed when thestrains were cultured in a medium containing sugars as carbon sources,and the expression was induced when the strains were cultured in amedium containing a non-sugar as carbon sources.

The cell extracts described above were analyzed by SDS-polyacrylamidegel electrophoresis (SDS-PAGE) according to the method of Laemli,Nature, 227, 680 (1970). The cell extract containing 15 μg of proteinwas put on 10% acrylamide gel. After electrophoresis, the gel wasstained with a staining solution (0.1% Coumassie Blue R250, 50%methanol) and then decolored with a decoloring solution (40% methanol,10% acetic acid).

By observation of the stained protein, it was confirmed with all thecoryneform bacteria described above except Corynebacterium callunae ATCC15991, that ICL protein having a size of about 48 kilodalton (kDa) waspresent in marked quantities in the cells cultured in MAYE medium. Inthe case of Corynebacterium callunae ATCC 15991, marked amounts of ICLprotein was also noted only in the cells cultured in MAYE medium, butthe size of the protein was about 52 kDa. In both of the strains,however, the ICL protein of 48 kDa or 52 kDa was barely or notdetectable in the cells cultured in MSYE medium.

In order to determine the proportion of the formed ICL protein of 48 kDaor 52 kDa to the total cell protein, the stained gel was scanned in onedirection using a one-dimensional densitometer (manufactured by ShimadzuSeisakusho Co., Model UV 265) and the distribution of color density wasmeasured in terms of visible absorption (560 nm). The results revealedthat the ICL protein of 48 kDa or 52 kDa amounted to the quantitycorresponding to 5 to 10% of the total cell protein in the cellscultured in MAYE medium. The presence of these proteins was hardlydetected in the cells cultured in MSYE medium.

In addition, the aforesaid cell extracts were analyzed by the Westernblotting method of Towbin, H., Proc. Natl. Acad. Sci. U.S.A., 76, 4350(1979) using an antibody to ICL protein of Corynebacterium glutamicumATCC 13032. Each of cell extract of the various coryneform bacteriaprepared above was put on SDS-polyacrylamide gel. After electrophoresis,a membrane filter (manufactured by ATO Co., Clear Blotting P Membrane)soaked in blotting buffer [25 mM Tris-HCl, 192 mM glycine (pH 8.3)] wasput on the gel. The filter was then inserted between filter papers(manufactured by Watman Co., 3 MM) soaked in the same blotting bufferand applied to a transcription device (manufactured by ATO Co.),followed by transcription at a constant current of 180 mA for one hour.After transcription, the filter was immersed in TBS buffer solution [20mM Tris-HCl, 0.5M NaCl (pH 7.5)] containing 1% BSA (bovine serumalbumin) and allowed to stand at room temperature for one hour.

Corynebacterium glutamicum ATCC 13032 also was grown in MAYE medium andthe extract of the obtained cells was put on SDS-polyacrylamide gel.After electrophoresis, the band corresponding to the 48 kDa protein wascut out of the gel and suspended in TBS buffer solution containing 0.05%Tween 20. The supernatant of the suspension was taken in portions andinjected into a mouse to prepare a polyclonal antibody (antibody to the48 kDa protein).

The filter subjected to transcription as described above was immersed inTBS buffer solution containing the 48 kDa protein antibody and 1% BSA,and allowed to stand at 4° C. overnight. Then, the filter was washedthree times with TBS buffer solution containing 0.05% Tween 20 andimmersed in TBS buffer solution containing anti-mouse IgG-peroxidase(manufactured by DACO Co.) and 1% BSA with shaking at room temperature.After one hour, the filter was washed with TBS buffer solutioncontaining 0.05% Tween 20. The filter was then immersed in a mixture ofa solution of 60 mg of 4-chloro-1-naphthol (manufactured by BIO-RAD Co.)in 20 ml of methanol and 100 ml of TBS buffer solution containing 60 μlof hydrogen peroxide to effect a color-developing reaction. The 48 kDaprotein antibody reacted not only with the ICL protein of 48 kDa fromCorynebacterium glutamicum ATCC 13032 but also with the ICL protein of48 kDa from Corynebacterium acetoacidophilum ATCC 13870, Corynebacteriumherculis ATCC 13868, Corynebacterium lilium ATCC 15990, Brevibacteriumimmariophilum ATCC 14068, Brevibacterium divaricatum ATCC 14020,Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum ATCC13655 and Microbacterium ammoniaphilum ATCC 15354, and with the ICLprotein of 52 kDa from Corynebacterium callunae ATCC 15991. From theforegoing, it was concluded that these proteins were identical orextremely similar to each other.

EXAMPLE 2 Cloning of ICL Gene of Corynebacterium glutamicum ATCC 13032

(1) Determination of N-terminal amino acid sequence of ICL protein

Proteins other than the ICL protein are hardly detected at about 48 kDain the cell extract of Corynebacterium glutamicum ATCC 13032. The ICLprotein of 48 kDa was isolated by SDS-PAGE, and the N-terminal aminoacid sequence was determined.

In a manner similar to Example 1, the cell extract of Corynebacteriumglutamicum ATCC 13032 cultured in MAYE medium was prepared and 3 μl ofthe extract was subjected to SDS-PAGE. After electrophoresis, the gelwas immersed in a buffer for transcription [10 mM3-cyclohexylamino-1-propanesulfonic acid, 10% methanol (pH 11.0)] atroom temperature for 5 minutes. The protein on the gel was transcribedonto PVDF membrane (manufactured by Millipore Co., 0.45 μm in pore size)soaked in methanol according to the method of Towbin et al., Proc. Natl.Acad. Sci. U.S.A., 76, 4530 (1979). After being washed with deionizedwater for 5 minutes, the PVDF membrane was stained with Coumassiestaining solution (0.1% Coumassie Blue R250, 50% methanol) for 5 minutesand then immersed in a decoloring solution (40% methanol, 10% aceticacid) for 5 minutes for decoloration. The PVDF membrane was thenimmersed in deionized water for 5 minutes for washing followed byair-drying. The ICL protein of 48 kDa stained on the membrane was cutout and the N-terminal amino acid sequence was determined according tothe method of Matsudaira et al., J. Biol. Chem., 262, 10035 (1987).

That is, the ICL protein transcribed on the membrane was subjected toEdman degradation using a protein sequencer (manufactured by AppliedBiosystems Co., Model 470) to analyze the N-terminal amino acid sequenceof the protein. The amino acid sequence was determined to be as shown bySeq. ID NO:1.

(2) Synthesis of oligonucleotide probe

An oligonucleotide having the nucleotide sequence (Seq. ID NO:2)corresponding to the amino acid sequence determined as above wassynthesized by the phosphoramidite method [M. H. Caruther et al.,Chemical Synthesis of Gene Fragments, a Laboratory Manual, Verlag Chemie(1982)] using an oligonucleotide synthesizer (manufactured by AppliedBiosystems Co., Model 380A).

This 50-mer oligonucleotide probe was 5'-labeled using [γ³² ] ATP(Amersham 3000 Ci/mmol) in the following manner. To 15 μl of kinasebuffer solution [50 mM Tris-HCl, 10 mM MgCl₂, 5 mM DTT, 0.1 mM EDTA (pH7.6)] were added 0.2 μg of the probe DNA and [γ³² ] ATP (150 μCi). Then,10 units of T4 polynucleotide kinase (manufactured by Takara Shuzo Co.,Ltd.) was added to the mixture and the reaction was carried out at 37°C. for 30 minutes. After phenol extraction, the reaction mixture wassubjected to gel filtration using Sephadex G50 to obtain the 5'-labeledprobe.

(3) Cloning of ICL gene-carrying fragment by colony hybridization

The seed culture (0.8 ml) of Corynebacterium glutamicum ATCC 13032cultured in NB medium was inoculated into 40 ml of SSM medium containing20 g of glucose, 10 g of (NH₄)₂ SO₄, 3 g of urea, 1 g of yeast extract,1 g of KH₂ PO₄, 0.4 g of MgSO₄.7H₂ O, 2 mg of FeSO₄.7H₂ O, 2 mg ofMnSO₄.4H₂ O, 60 μg of biotin, 2 mg of thiamine hydrochloride and 50 mgof NaCl in 1 liter of water and adjusted to pH 7.2, followed by shakeculture at 30° C. The optical density (OD) was measured at 660 nm with aHitachi Colorimeter (Model 100-20), and when the OD reached 0.2,penicillin G was added to the culture at a concentration of 0.5 unit/ml.Culturing was continued until the OD reached 0.6. Then, the cells werecollected from the culture, washed with TES buffer solution [0.03MTris-HCl, 0.005M EDTA, 0.05M NaCl (pH 8.0)], and suspended in 10 ml oflysozyme solution [25% sucrose, 0.1M NaCl, 0.05M Tris-HCl, 0.8 mg/mllysozyme (pH 8.0)]. The suspension was kept at 37° C. for 2 hours. Fromthe collected cells, a high molecular weight chromosomal DNA wasisolated according to the method of Saito et al., Biochem. Biophys.Acta, 72, 619 (1963). On the other hand, pUC19 was prepared from E. coliATCC 33694 carrying pUC19 (manufactured by Takara Shuzo Co., Ltd.) in aconventional manner according to the method of Birnboim et al., NucleicAcids Res., 7, 1513 (1979).

Twenty units of Hind III was added to 98 μl of buffer solution B [10 mMTris-HCl (pH 7.5), 50 mM NaCl, 10 mM MgCl₂ 1 mM DTT] containing 5 μg ofthe chromosomal DNA obtained from Corynebacterium glutamicum ATCC 13032.The reaction was carried out at 37° C. for 2 hours. On the other hand, 5units of Hind III was added to 48.5 μl of buffer solution B containing 1μg of pUC19 plasmid DNA and the reaction was carried out at 37° C. forone hour. After these reaction products were mixed, phenol extractionand ethanol precipitation were carried out to recover DNAs. All of therecovered DNA was dissolved in 59 μl of ligation buffer solution [20 mMTris-HCl (pH 7.6), 10 mM MgCl₂, 10 mM DTT, 1 mM ATP], and 350 units ofT4 ligase was added to the solution. The ligation reaction was carriedout at 16° C. for 15 hours.

E. coli ATCC 33694 was transformed using this DNA reaction mixture bythe method of Dagert et al., Gene, 6, 23 (1979). An LB plate [1%trypton, 0.5% yeast extract, 0.5% NaCl (pH 7.4)] containing 100 μg/mlampicillin was covered with a nitrocellulose filter (manufactured byGelman Science Co., Bio Trace™NT) and the transformants were applied tothe surface of the filter. After the plate was allowed to stand at 37°C. for 16 hours, colonies formed on the filter were replicated on twonitrocellulose filters. The three filters were transferred to an LBplate containing 100 μg/ml ampicillin and allowed to stand at 37° C. for6 hours for proliferation. Two of the replicated nitrocellulose filterswere transferred to an LB plate containing 250 μg/ml chloramphenicol and100 μg/ml ampicillin. After culturing at 37° C. for 16 hours, thefilters were transferred successively onto Watman 3 MM filter paperrespectively soaked in 0.5M NaOH solution, 1.0M Tris-HCl (pH 7.5), 1.5MNaCl-0.5M Tris-HCl (pH 7.5) solution and 2×SSC solution [0.3M NaCl,0.03M Na₃ -citrate (pH 7.0)] to expose and denature the DNAs from thecolonies. After air-drying, DNAs were immobilized on the filters byheating at 80° C. for 3 hours. On the other hand, the third filter waskept on the plate and stored at 4° C.

The replica filter on which the gene library had been immobilized wasimmersed in 3×SSC solution [0.45M NaCl, 0.045M Na₃ -citrate (pH 7.0)] at65° C. for 30 minutes. Then, the filter was transferred into 1×Denhardtsolution (0.2% Ficoll, 0.2% polyvinylpyrrolidone, 0.2% BSA) and allowedto stand at 65° C. for one hour. The filter was put in a polypropylenebag charged with pre-hybridization buffer solution [1×Denhardt solution,1M NaCl, 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.1% SDS, 100 μg/mldenatured salmon sperm DNA] and pretreated at 65° C. for 3 hours. Then,0.2 μg of the radio isotope-labeled 50-mer oligonucleotide probe ofExample 2(2) was added and hybridization was carried out at 40° C. for16 hours. The filters were washed successively with 6×SSC solution [0.9M NaCl, 0.09M Na₃ -citrate (pH 7.0)] twice at 4° C. for 5 minutes,twice at 52° C. for 30 minutes and twice at 4° C. for 5 minutes. Afterair-drying, each filter was brought into contact with an X ray film(manufactured by Fuji Photo Film Co., Ltd.) and exposed to light.

One colony hybridized to the probe out of about 8500 clones. A colonycorresponding to this hybridized colony was isolated from the storedplate and a clone of the colony was tested. As the result, it was foundthat the clone had a structure in which a Hind III fragment of 6.0 kbhad been inserted in the Hind III site of pUC19. This plasmid was namedpKT4.

EXAMPLE 3 Expression of ICL Gene in ICL Gene-Amplified Strain

(1) Expression of ICL gene of Corynebacterium glutamicum ATCC 13032(pKT10)

In order to confirm that the ICL gene is carried on the cloned fragmentdescribed above, pKT4 was inserted into a vector for coryneformbacteria, pCG116 (Japanese Published Unexamined Patent Application No.265892/89). Plasmid pCG116 was isolated from the cultured cells ofCorynebacterium glutamicum ATCC 13032 carrying pCG116 according to thefollowing method. The seed culture (8 ml) of pCG116-carryingCorynebacterium glutamicum ATCC 13032 grown in NB medium containing 100μg/ml spectinomycin was inoculated into 400 ml of SSM medium containing100 μg/ml spectinomycin, followed by shake culture at 30° C. When the ODreached 0.2, penicillin G was added to the culture at a concentration of0.5 unit/ml. Culturing was continued until the OD reached 0.6, and thenthe cells were collected from the culture. After washing with TES buffersolution, the cells were suspended in 10 ml of lysozyme solution andsubjected to reaction at 37° C. for 4 hours. To the reaction mixturewere successively added 2.4 ml of 5M NaCl, 0.6 ml of 0.5M EDTA (pH 8.5),and 4.4 ml of a solution comprising 4% sodium lauryl sulfate and 0.7MNaCl. After gentle mixing, the mixture was allowed to stand on ice waterfor 15 minutes. The resulting lysate was transferred to a centrifugetube and centrifuged at 4° C. for 60 minutes at 69,400×g to recover thesupernatant. To the supernatant was added polyethylene glycol (PEG 6000)in an amount corresponding to 10% by weight, followed by gentle mixing.The resulting solution was put on ice water, and after 10 hours, it wascentrifuged for 10 minutes at 1,500×g to recover pellets. TES buffersolution (5 ml) was added to dissolve the pellets, followed by additionof 2.0 ml of 1.5 mg/ml ethidium bromide. Then, 7.5 g of cesium chloridewas added thereto and gently dissolved, and the density was adjusted to1580. The resulting solution was ultra-centrifuged at 18° C. for 48hours at 105,000×g. A high density band located at the lower part of thecentrifuge tube, which was detected under UV irradiation, was taken witha syringe from the side of the centrifuge tube, thereby isolating pCG116plasmid DNA. The fraction was treated five times with an equal amount ofisopropyl alcohol solution (by volume, 90% isopropyl alcohol, 10% TESbuffer solution) to extract and remove ethidium bromide. Thereafter,dialysis was carried out against TES buffer solution.

Five units of Bam HI was added to 19 μl of buffer solution C [10 mMTris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl₂, 1 mM DTT] containing 1 μgof pCG116 plasmid DNA, and the reaction was carried out at 37° C. forone hour. On the other hand, 5 units of Bam HI was added to 49 μl ofbuffer solution C containing 1 μg of pKT4 plasmid DNA isolated from thecultured cells of E. coli ATCC 33694 transformants by the process usedin Example 2(3), and the reaction was carried out at 37° C. for onehour. Both reaction mixtures were subjected to 0.8% agarose gelelectrophoresis, and a fragment of 6.5 kb and a fragment of 8.7 kb wererespectively recovered using a kit for recovery and purification of DNA(manufactured by Asahi Glass Co., Ltd.). The DNA fragments were ligatedwith each other by conventional ligase treatment. E. coli ATCC 33694 wastransformed using this ligase reaction mixture according to the methoddescribed in Example 2(3), and spectinomycin-resistant transformantswere isolated on an LB plate containing 25 μg of spectinomycin. PlasmidpKT10 shown in FIG. 2 was isolated from one of the transformants.

pKT10 DNA (1 μg) was used for protoplast transformation ofCorynebacterium glutamicum ATCC 13032 [J. Bacteriol., 159, 306 (1984)].The protoplasts were prepared in the following manner. A seed culture(0.8 ml) of ATCC 13032 strain grown in NB medium was inoculated into 40ml of SSM medium, followed by shake culture. When the OD reached 0.2,penicillin G was added to the culture at a concentration of 0.5 unit/ml.Culturing was continued until the OD reached 0.6. Then, the cells werecollected from the culture and suspended in 10 ml of RCGP medium [mediumcontaining 5 g of glucose, 5 g of Casamino acid, 2.5 g of yeast extract,3.5 g of K₂ HPO₄, 1.5 g of KH₂ PO₄, 0.41 g of MgCl₂.6H₂ O, 10 mg ofFeSO₄.7H₂ O, 2 mg of MnSO₄.4-6H₂ O, 0.9 mg of ZnSO₄.7H₂ O, 0.04 mg of(NH₄)₆ Mn₇ O₂₄.4H₂ O, 30 μg of biotin, 2 mg of thiamine hydrochloride,135 g of sodium succinate and 30 g of polyvinylpyrrolidone (molecularweight; 10000) in 1 liter of water and adjusted to pH 7.4] containing 1mg/ml lysozyme and adjusted to pH 7.6. The suspension was allowed tostand at 30° C. for 16 hours to obtain a protoplast suspension. Theprotoplast suspension was centrifuged at 2,500×g for 5 minutes and theprecipitated protoplasts were suspended in 1 ml of TSMC buffer solution[10 mM MgCl₂, 30 mM CaCl₂, 50 mM Tris-HCl, 400 mM sucrose (pH 7.5)],followed by centrifugation and washing. The resulting protoplasts wereresuspended in 0.1 ml of TSMC buffer solution, and the suspension wasmixed with 10 μl of a solution containing pKT10 plasmid DNA preparedabove, followed by addition of 0.8 ml of TSMC buffer solution containing20% PEG6000. The resulting mixture was allowed to stand in ice water for20 minutes and then at 37° C. for 3 minutes and centrifuged at 2,500×gfor 5 minutes to remove the supernatant. The precipitated protoplastswere suspended in 1 ml of RCGP medium, and 0.2 ml of the suspension wassmeared on RCGP plate containing 400 μg/ml spectinomycin. Incubation wascarried out at 30° C. for 7 days to obtain transformants. The plasmidcontained in the transformants was analyzed by digestion withrestriction enzymes, whereby it was confirmed that the transformantscarried pKT10.

In a similar manner, an ICL-deficient mutant acquired as an aceticacid-non-assimilative mutant from Corynebacterium glutamicum ATCC 13032according to a known process [J. Appl. Microbiol., 15, 27 (1969)] wastransformed using pKT10 plasmid DNA solution. The ICL-deficient mutantcarrying pKT10 introduced by the transformation acquired the ability ofgrowth in MA medium containing acetic acid as a carbon source [mediumcontaining 20 g of ammonium acetate, 10 g of (NH₄)₂ SO₄, 3 g of urea, 1g of KH₂ PO₄, 0.4 g of MgSO₄.7H₂ O, 2 mg of FeSO₄.7H₂ O, 2 mg ofMnSO₄.4H₂ O, 60 μg of biotin, 2 mg of thiamine hydrochloride and 50 mgof NaCl in 1 liter of water and adjusted to pH 7.2] and had ICL activitycomparable to ATCC 13032 (pKT10) strain. It was confirmed therefrom thatthe Hind III DNA fragment of 6.0 kb obtained from Corynebacteriumglutamicum ATCC 13032 which was present on pKT10 contained the ICL gene.

Corynebacterium glutamicum ATCC 13032 and ATCC 13032 (pKT10) werecultured at 30° C. for 16 hours in MSYE medium containing sucrose as acarbon source and in MAYE medium containing acetic acid as a carbonsource. After the cells were collected, the cell extract was prepared.

The ICL activity of the cell extract was determined by the methoddescribed in Example 1. The results are shown in Table 1. The cells ofATCC 13032 (pKT10) cultured in MAYE medium showed a high level of ICLactivity, as was also the case with ATCC 13032. However, the activitylevel of ATCC 13032 (pKT10) was about six times higher than that of ATCC13032.

The cell extracts of the two strains were analyzed by SDS-polyacrylamidegel electrophoresis, whereby large quantities of the ICL protein of 48kDa were detected only in the cells cultured in MAYE medium. The amountof the ICL protein of 48 kDa produced by Corynebacterium glutamicum ATCC13032 (pKT10) was about 33% of the total cell protein, which was 5 to 6times larger than that of ATCC 13032 strain. The foregoing resultsreveal that the expression of ICL gene was regulated also when the copynumber was increased.

(2) Expression of amplified ICL gene in other hosts

pKT10 was introduced into 9 strains of coryneform bacteria shown inTable 1 in a similar manner as in Example 3(1). Additionally, pKT10 wasintroduced into Brevibacterium ammoniagenes ATCC 6872 by the methoddescribed in Japanese Published Unexamined Patent Application No.185372/88. That is, 0.8 ml of the seed culture of this strain culturedin NB medium was inoculated into 40 ml of GIII medium containing 15 g ofglucose, 8 g of (NH₄)₂ SO₄, 1.2 g of urea, 1.2 g of yeast extract, 0.5 gof KH₂ PO₄, 0.5 g of K₂ HPO₄, 0.1 g of MgSO₄.7H₂ O, 2 mg of FeSO₄.7H₂ O,1 mg of ZnSO₄.7H₂ O , 1 mg of MnSO₄.4-6H₂ O, 0.1 mg of biotin, 2 mg ofthiamine hydrochloride, 10 mg of calcium panthothenate, 100 mg ofadenine and 100 mg of guanine in 1 liter of water and adjusted to pH7.2, followed by shake culture at 30° C. Penicillin G was added to theculture at a concentration of 0.3 unit/ml at the initial stage oflogarithmic growth phase (cell concentration: 10⁸ cells/ml). Culturingwas continued for an additional 3 hours, and then the culture wascentrifuged at 3,000 rpm for 10 minutes to recover the cells. Afterwashing with GIII medium, the cells were suspended in 10 ml of P3hypertonic solution [70 mM NaCl, 5 mM MgCl₂, 5 mM CaCl₂, 25 mM Tris-HCl,1.6M D-sorbitol (pH 7 6)] containing 2.0 mg/ml lysozyme and 0.6 mg/mlachromopeptidase. The suspension was allowed to stand at 30° C. for 16hours to prepare protoplasts. Transformants were obtained using the thusprepared protoplasts according to the method of Example 3(1).

The obtained transformants, except those of Brevibacterium ammoniagenesATCC 6872, were cultured in MSYE medium and MAYE medium and the ICLactivity of the cell extracts was determined. The ICL activity ofBrevibacterium ammoniagenes ATCC 6872 and ATCC 6872 (pKT10) wasdetermined using the cell extract of the cells cultured in MSYE mediumand the cell extract of the cells obtained by culturing the cells inMSYE medium, suspending the cultured cells in MAYE medium and thenincubating the suspension at 30° C. for 16 hours. As shown in Table 1,ICL activity at high levels was detected in the cells cultured in MAYEmedium with all of the pKT10 transformants, as in the case ofCorynebacterium glutamicum ATCC 13032 (pKT10).

                  TABLE 1                                                         ______________________________________                                                         ICL Specific Activity                                                         (U/mg protein)                                                                  MSYE      MAYE                                             Strain             Medium    Medium                                           ______________________________________                                        Corynebacterium glutamicum                                                    ATCC 13032         ND         760                                             ATCC 13032 (pKT10) ND        4670                                             Corynebacterium acetoacidophilum                                              ATCC 13870         ND         980                                             ATCC 13870 (pKT10) 170       6050                                             Corynebacterium callunae                                                      ATCC 15991         ND         480                                             ATCC 15991 (pKT10) ND        4330                                             Corynebacterium herculis                                                      ATCC 13868         ND         350                                             ATCC 13868 (pKT10) 160       1830                                             Corynebacterium lilium                                                        ATCC 15990         ND         300                                             ATCC 15990 (pKT10) 170       2140                                             Brevibacterium immariophilum                                                  ATCC 14068         ND         130                                             ATCC 14068 (pKT10) 190       6690                                             Brevibacterium divaricatum                                                    ATCC 14020         ND         430                                             ATCC 14020 (pKT10) 140       3980                                             Brevibacterium flavum                                                         ATCC 14067         ND         550                                             ATCC 14067 (pKT10) 120       2940                                             Brevibacterium lactofermentum                                                 ATCC 13655         ND         230                                             ATCC 13655 (pKT10) 120       2760                                             Microbacterium ammoniaphilum                                                  ATCC 15354         ND         450                                             ATCC 15354 (pKT10)   50      4000                                             Brevibacterium ammoniagenes                                                   ATCC 6872          ND        ND                                               ATCC 6872 (pKT10)  ND        1730                                             ______________________________________                                         ND: not detected                                                         

EXAMPLE 4 Analysis of ICL Gene-Carrying Fragment

(1) Restriction map of DNA fragment

pKT4 plasmid DNA (1 μg) was treated with 10 to 12 units of restrictionenzymes (Afl II, Alu I, Bgl II, Cla I, Hind III, Hpa I, Nco I, Nru I,Sma I, Sph I, Stu I, Xho I), alone or in combination, at 37° C. (enzymesother than Sma I) or at 30° C. (Sma I) for one hour. The reactionmixture was subjected to 0.8% agarose gel electrophoresis or 5%polyacrylamide gel electrophoresis. Measurement of the size of thefragments formed revealed that the cloned Hind III DNA fragment of 6.0kb had a structure shown by the restriction map of FIG. 1.

(2) Subcloning

In order to locate the ICL gene cloned on the 6.0 kb Hind III DNAfragment, several regions were subcloned. Ten units each of Hind III andXho I were added to 48 μl of buffer solution C containing 2 μg of pKT4plasmid, and the reaction was carried out at 37° C. for 2 hours. On theother hand, 10 units each of Hind III and Xho I were added to 18 μl ofbuffer solution C containing 2 μg of pUC19, and the reaction was carriedout at 37° C. for 2 hours. Both reaction mixtures were subjected to 0.8%agarose gel electrophoresis, and a fragment of 1.9 kb and a fragment of2.7 kb were respectively recovered using a kit for recovery andpurification of DNA. The two DNA fragments were ligated with each otherby ligase treatment in conventional manner. The ligase reaction mixturewas used for transformation of E. coli ATCC 33694 and plasmid pKT5 wasobtained. pKT5 DNA was then digested with Kpn I and ligated with pCG116plasmid DNA, which is a vector for Corynebacterium glutamicum, digestedwith the same restriction enzyme to prepare plasmid pKT13. In addition,2.2 kb Sma I-Bgl II fragment, 2.1 kb Hpa I-Bgl II fragment and 1.2 kbStu I-Bgl II fragment were obtained from pKT4 and then inserted into SmaI-Bam HI linker site of pCG116 to prepare plasmids pKT19, pKT20 andpKT21, respectively. The DNA fragments cloned on these plasmids areshown in FIG. 3.

Corynebacterium glutamicum ATCC 13032 was transformed with these plasmidDNAs. The transformants were cultured in MAYE medium and the ICLactivity of the cultured cells was determined (FIG. 3). ThepKT19-carrying strain and the pKT20-carrying strain showed an activitylevel as high as that of the pKT10-carrying strain, whereas thepKT13-carrying strain and the pKT21-carrying strain only gave theactivity at almost the same level as that of the host. On the basis ofthese results, the ICL gene was to be located on the 2.1 kb Hpa I-Bgl IIDNA fragment shown by an arrow in the upper part of FIG. 3.

(3) Nucleotide sequence of the region of ICL gene

The Hpa I-Bgl II DNA fragment of 2.1 kb encoding ICL was digested at itsrestriction enzyme sites and inserted into plasmids pUC118 and pUC119(manufactured by Takara Shuzo Co., Ltd.) digested with the correspondingrestriction enzymes. By using the thus prepared plasmids, the nucleotidesequence of the fragment encoding ICL was determined according to amodification of the M13 chain termination method by Messing et al.,Methods in Enzymology, 101, 20 (1983). The result is shown by the DNAnucleotide sequence and the amino acid sequence corresponding to the ICLstructural gene represented by Seq. ID NO:3. The nucleotide sequence wasfound to contain the open reading frame (1293 bp) comprising 431 aminoacid residues including the sequence corresponding to codons for 17amino acids out of the N-terminal 18 amino acid residues shown inExample 2(1). This indicates that ICL promoter activity is attributableto the DNA sequence upstream of ATG. At the position downstream of stopcodon TAG by 27 bp, a sequence considered to function in the terminationof transcription was present at positions 1833 to 1846 and 1850 to 1863of the DNA nucleotide sequence shown by Seq. ID NO:3.

EXAMPLE 5 Homology of ICL Genes of Coryneform bacteria

Homology of chromosomal DNA fragments of various coryneform bacteria wasexamined according to the Southern hybridization method of Read et al.,Nucleic Acid Res., 13, 7207 (1985) using as a probe the 50-meroligonucleotide corresponding to the N-terminal amino acid sequence ofICL described in Example 2(2) or an Hpa I-Afl II fragment of 0.5 kb(Seq. ID NO: 3) which is 5'-untranslated region.

In order to prepare the Hpa I-Afl II fragment of 0.5 kb, 10 units of AflII was added to 49 μl of buffer solution E [10 mM Tris-HCl (pH 7.5), 40mM KCl, 10 mM MgCl₂, 1 mM DTT] containing 2 μg of plasmid pKT10 (FIGS. 2and 3) and the reaction was carried out at 37° C. for one hour. Then, 3μl of 1M KCl and 10 units of Hpa I were further added to the mixture andthe reaction was carried out at 37° C. for one hour. The reactionmixture was subjected to 1.2% agarose gel electrophoresis and the 0.5 kbHpa I-Afl II fragment was recovered using a kit for recovery andpurification of DNA.

The 50-mer oligonucleotide was labeled at the 5' end according to themethod of Example 2(2). The 0.5 kb Hpa I-Afl II fragment was labeledwith [³² P] using Nick Translation Kit (manufactured by Takara ShuzoCo., Ltd.).

Chromosomal DNAs were prepared from Corynebacterium glutamicum ATCC13032, Corynebacterium acetoacidophilum ATCC 13870, Corynebacteriumcallunae ATCC 15991, Corynebacterium herculis ATCC 13868, Brevibacteriumdivaricatum ATCC 14020, Brevibacterium lactofermentum ATCC 13655 andMicrobacterium ammoniaphilum ATCC 15354 respectively according to themethod described in Example 2(3). To 98 μl of buffer solution Bcontaining 5 82 g of each chromosomal DNA was added 20 units of HindIII, and the reaction was carried out at 37° C. for 2 hours. Tenmicroliters each of these reaction mixtures were respectively subjectedto 0.8% agarose gel electrophoresis. After the electrophoresis, the gelwas immersed in 0.25M HCl and shaken for 15 minutes. Then, the gel wasrinsed with deionized water and put on a filter paper (Watman 3 MM)soaked in 0.4M NaCl. A nylon filter (manufactured by BIO-RAD Co., Ltd.,Zeta Probe Membrane), a filter paper and an appropriate weight weresuccessively layered on the gel and 0.4M NaOH was provided from the backof the gel through the filter paper to transfer DNA onto the nylonfilter. The filter was washed with 6×SSC, air-dried and subjected to ahybridization test. The filter was immersed in 20 ml of aprehybridization solution (6× SSC, 0.01M EDTA, 1% Ficoll, 1%polyvinylpyrrolidone, 1% bovine serum albumin, 0.5% SDS, 0.1 mg/mldenatured salmon sperm DNA) and heated at 68° C. for 3 hours. Then, thefilter was transferred to a hybridization solution obtained by adding0.2 μg of each labeled probe to 20 ml of the prehybridization solution.When the 50-mer oligonucleotide was used as a probe, hybridization wascarried out at 40° C. for 16 hours. In the case of the 0.5 kb Hpa I-AflII fragment, hybridization was carried out at 68° C. for 16 hours. Thetreated filter was then immersed in SWS (0.3×SSC, 0.05% SDS). When the50-mer oligonucleotide was used as a probe, treatment at 52° C. for 30minutes was carried out twice; and when the 0.5 kb Hpa I-Afl II fragmentwas used as a probe, treatment at 68° C. for 30 minutes was carried outtwice. After washing, each filter was air-dried, brought into contactwith an X ray film (manufactured by Fuji Photo Film Co., Ltd.) andexposed to light.

In the case of the coryneform bacteria other than Corynebacteriumcallunae ATCC 15991, hybrids with Hind III chromosomal DNA fragments ofabout 6.0 kb were formed by using either probe. On the other hand, inthe case of Corynebacterium callunae ATCC 15991, hybrids with Hind IIIchromosomal DNA fragments of about 2.0 kb were formed by using eitherprobe. These results reveal that the ICL genes of coryneform bacteriahave homology to that of Corynebacterium glutamicum ATCC 13032 not onlyin the ICL structural gene region but also in the promoter region.

EXAMPLE 6 Expression of Chloramphenicol Acetyltransferase StructuralGene by the ICL Promoter

Plasmid pKK232-8 of E. coli carries a DNA fragment which contains in itsstructure: (1) the region from the sequence necessary for translationinitiation to the structural gene in the E. coli-derived chloramphenicolacetyltransferase gene region lacking the promoter sequence; and (2)located downstream of the region, terminator T₁ T₂ derived from E. coliribosome RNA gene [Brosius, J., Gene, 27, 151 (1984)]. Plasmid pCGKK27-3containing the DNA fragment and capable of replicating in coryneformbacteria was prepared as shown in FIG. 4.

To 50 μl of buffer solution C containing 5 μg of pKK232-8 (manufacturedby Pharmacia Fine Chemicals) was added 0.3 unit of Pst I. Aftertreatment at 37° C. for one hour, the mixture was heated at 68° C. for10 minutes to terminate the reaction. On the other hand, 2 μg of pCG11,a vector for coryneform bacteria (Japanese Published Unexamined PatentApplication No. 134500/82), prepared from a pCG11-carrying strain by themethod described in Japanese Published Unexamined Patent Application No.186489/82 was treated with 12 units of Pst I in 49 μl of buffer solutionC at 37° C. for one hour, followed by heating. The reaction mixtures ofpKK232-8 and pCG11 were subjected to 0.8% agarose gel electrophoresis,and a DNA fragment of 5.1 kb and a DNA fragment of 6.8 kb wererespectively recovered using a kit for recovery and purification of DNA.The DNA fragments were mixed with each other and T4 ligase was added tothe mixture to cause ligation.

E. coli ATCC 33694 was transformed using this ligase reaction mixtureaccording to the method described in Example 2(3), andspectinomycin-resistant transformants were isolated on an LB platecontaining 25 μg/ml spectinomycin. Plasmid DNAs extracted from thetransformants were analyzed by digestion with restriction enzymes. Fromone of the transformants, plasmid pCGKK27-3 carrying pKK232-8 ligatedwith pCG11 was obtained as shown in FIG. 4.

Corynebacterium glutamicum ATCC 13032 was transformed with pCGKK27-3 bythe method described in Example 3(1). It was confirmed that thetransformant selected on the basis of spectinomycin resistance carriedpCGKK27-3 but did not show chloramphenicol resistance and thechloramphenicol acetyltransferase structural gene was not expressed inATCC 13032.

A DNA fragment having ICL promoter activity was inserted into pCGKK27-3(cf. FIG. 5). As the fragment containing the ICL promoter, the Sma I-AluI fragment of 0.6 kb shown in FIG. 3 was used. pKT19 (5 μg) was treatedwith 10 units of Sma I in 49 μl of buffer solution D [10 mM Tris-HCl (pH7.5), 20 mM KCl, 10 mM MgCl₂, 1 mM DTT] at 30° C. for one hour. Then, 6μl of 0.2M KCl and 10 units of All II were added to the reaction mixturefollowed by reaction at 37° C. for one hour. The reaction mixture wassubjected to 1% agarose gel electrophoresis, and a Sma I-All II DNAfragment of 0.8 kb was isolated using a kit for recovery andpurification of DNA. To 49 μl of buffer solution A [10 mM Tris-HCl (pH7.5), 10 mM MgCl₂, 1 mM DTT] containing this DNA was added 10 units ofAlu I. The reaction was carried out at 37° C. for one hour, followed byheating at 68° C. for 10 minutes to prepare a solution containing theSma I-Alu I DNA fragment.

On the other hand, 5 μg of pCGKK27-3 isolated from the transformant ofCorynebacterium glutamicum ATCC 13032 was treated with 10 units of Sma Iin 49 μl of buffer solution D at 30° C. for one hour, followed by heattreatment.

The obtained solution containing pCGKK27-3 was mixed with the aforesaidsolution containing Sma I-Alu I DNA fragment, followed by treatment withligase in conventional manner. Corynebacterium glutamicum ATCC 3032 wastransformed with this ligase reaction mixture, and the cell suspensionwas smeared on RCGP plate containing 10 mg/ml ammonium acetate, 400μg/ml spectinomycin and 5 μg/ml chloramphenicol. Incubation was carriedout at 30° C. for 7 days to obtain colonies of the transformants. Fromone of the transformants, plasmid pKT22 was obtained, which had astructure wherein the Sma I-Alu I DNA fragment was inserted just beforethe DNA fragment containing the chloramphenicol acetyltransferasestructural gene (see FIG. 5).

This transformant and ATCC 13032 strain were each cultured in MSYEmedium and MAYE medium at 30° C. for 16 hours. The cultured cells werecollected and disrupted, and the obtained cell extracts were examinedfor chloramphenicol acetyltransferase activity by the method of Shaw etal., Methods in Enzymology, 43, 737 (1975). The activity was indicatedas U/mg protein, one unit being defined as that enzymatic activity whichcatalyzes the acetylation of 1 μmol of chloramphenicol in one minute.

pKT22 was introduced into the coryneform bacteria shown in Table 2 in asimilar manner as above. The obtained transformants were cultured inMSYE medium and MAYE medium and the chloramphenicol acetyltransferaseactivity of the cell extracts was determined. The results are shown inTable 2.

                  TABLE 2                                                         ______________________________________                                                          Chloramphenicol                                                               Acetyltransferase                                                             Specific Activity                                                             (U/mg protein)                                                                  MSYE     MAYE                                             Strain              Medium   Medium                                           ______________________________________                                        Corynebacterium glutamicum                                                    ATCC 13032          0        0                                                ATCC 13032 (pKT22)  0.6      27.4                                             Corynebacterium herculis                                                      ATCC 13868          0        0                                                ATCC 13868 (pKT22)  0.2      13.2                                             Brevibacterium divaricatum                                                    ATCC 14020          0        0                                                ATCC 14020 (pKT22)  0.5      25.8                                             Brevibacterium lactofermentum                                                 ATCC 13655          0        0                                                ATCC 13655 (pKT22)  0.3      22.0                                             Brevibacterium ammoniagenes                                                   ATCC 6872           0        0                                                ATCC 6872 (pKT22)   0.1      12.6                                             ______________________________________                                    

As shown in Table 2, the transformants produced chloramphenicolacetyltransferase in large amounts when cultured in MAYE medium. Markedamounts of chloramphenicol acetyltransferase were observed as a proteinband of 24 kDa in analysis by SDS-polyacrylamide gel electrophoresis.

From the foregoing results, it was confirmed that the expression of thechloramphenicol acetyltransferase structural gene was induced by the ICLpromoter.

EXAMPLE 7 Expression of β-Galactosidase Structural Gene by the ICLPromoter

An expression vector capable of expressing β-galactosidase structuralgene of E. coli by the ICL promoter was prepared according to the stepsoutlined in FIG. 6.

To 98 μl of buffer solution C containing 10 μg of plasmid pKT20 carryingthe ICL gene was added 24 units of Stu I. After reaction at 37° C. for 2hours, the reaction mixture was extracted once with an equal amount ofphenol. Further, extraction was carried out once with an equal amount ofchloroform/isoamyl alcohol (24/1, v/v), followed by ethanolprecipitation and vacuum drying. The obtained DNA was subjected to adeletion treatment with exonuclease III using a Kilo Sequence DeletionKit (manufactured by Takara Shuzo Co., Ltd.). Twenty units of Xho I wasadded to 40 N1 of buffer solution C containing the deletion fragments,and the reaction was carried out at 37° C. for 2 hours.

Separately, a DNA fragment containing the β-galactosidase structuralgene was prepared from plasmid pE'lac1 (Japanese Published UnexaminedPatent Application No. 273469/88). Plasmid pE'lac1 contains a DNAsequence wherein the region upstream of the codon for the 8th amino acidfrom the N-terminus of the β-galactosidase structural gene in thelactose operon of E. coli is deleted. To 49 μl of buffer solution Dcontaining 5 μg of pE'lac1 was added 10 units of Sma I, and the reactionwas carried out at 30° C. for 2 hours. Then, 1.5 μl of 5M NaCl and 10units of Sal I were added to the reaction mixture, and the reaction wascarried out at 37° C. for 2 hours. The digested fragments of plasmidspKT20 and pE'lac1 were separated by 0.8% agarose gel electrophoresis,and a fragment of about 7.6 kb and a fragment of about 6.2 kb wererespectively recovered using a kit for recovery and purification of DNA.The fragments were ligated with each other by ligase treatment inconventional manner.

Corynebacterium glutamicum ATCC 13032 was transformed using this ligasereaction mixture by the method of Example 3(1). Then, the cellsuspension was smeared on RCGP plate containing 400 μg/ml spectinomycin,40 μg/ml 5-bromo-4-chloro-3-indolyl-μ-D-galactoside (X-gal) and 0.01g/ml ammonium acetate. Incubation was carried out at 30° C. for 7 daysto obtain the transformants stained blue on the plate. One of thetransformants carried plasmid pKT23 in which the lactose operon-derivedDNA fragment of 6.2 kb was inserted into the DNA fragment carrying theICL gene (see FIG. 6).

This transformant and Corynebacterium glutamicum ATCC 13032 werecultured in MSYE medium and MAYE medium at 30° C. for 16 hours. Thecultured cells were collected and disrupted, and the cell extracts wereexamined for β-galactosidase activity according to the method of Milleret al., Experiments in Molecular Genetics, 352, Cold Spring HarborLaboratory (1972). The specific enzymatic activity per mg of protein wascalculated and indicated as U/mg protein, one unit being defined as thatenzymatic activity which catalyzes the formation of 1 μmol ofo-nitrophenol in one minute.

pKT23 was introduced into the coryneform bacteria shown in Table 3 in asimilar manner as above. The obtained transformants were cultured inMSYE medium and MAYE medium and the β-galactosidase activity of the cellextracts was determined. The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                                          β-galactosidase                                                          Specific Activity                                                             (U/mg protein)                                                                  MSYE     MAYE                                             Strain              Medium   Medium                                           ______________________________________                                        Corynebacterium glutamicum                                                    ATCC 13032           0         0                                              ATCC 13032 (pKT23)  700      30900                                            Corynebacterium herculis                                                      ATCC 13868           0         0                                              ATCC 13868 (pKT23)  300      14400                                            Brevibacterium divaricatum                                                    ATCC 14020           0         0                                              ATCC 14020 (pKT23)  500      27500                                            Brevibacterium lactofermentum                                                 ATCC 13655           0         0                                              ATCC 13655 (pKT23)  350      22700                                            ______________________________________                                    

As shown in the table, only the transformants produced β-galactosidasein large amounts when cultured in MAYE medium. Marked amounts ofβ-galactosidase were detected as a protein band of a little larger than116 kDa in the analysis by SDS-polyacrylamide gel electrophoresis.

The ligation site of the DNA fragment of the 5'-end region in the ICLgene region and the β-galactosidase structural gene on pKT23 wasexamined. To 49 μl of buffer solution A containing 2 μg of pKT23 and 2μg of plasmid pUC118 (manufactured by Takara Shuzo Co., Ltd.),respectively, was added 10 units of Kpn I, and the reaction was carriedout at 37° C. for 2 hours. Then, 0.5 μl of 5M NaCl and 10 units of BamHI were added to the reaction mixture and the volume of the mixture wasadjusted to 55 μl by addition of sterilized water. The reaction wascarried out at 37° C. for 2 hours. These digestion products of pKT23 andpUC118 were subjected to 0.8% agarose gel electrophoresis, and afragment of about 0.7 kb and a fragment of about 7.2 kb wererespectively recovered using a kit for recovery and purification of DNA.After the fragments were ligated by ligase treatment in conventionalmanner, the nucleotide sequence of the obtained DNA was determinedaccording to the method described in Example 4(3).

The result revealed that the β-galactosidase structural gene lackingN-terminal 8 amino acids was ligated in frame with the DNA fragmentencoding the 1st to 63rd amino acids from the N-terminus of isocitratelyase shown by Seq. ID NO:4.

From the foregoing results, it was confirmed that the synthesis ofβ-galactosidase-fused protein was induced under the control of the ICLpromoter.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:18 amino acid                                                      (B) TYPE:amino acid                                                           (C) STRANDEDNESS:single                                                       (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (v) FRAGMENT TYPE:N-terminal fragment                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        SerAsnValGlyLysProArgThrAlaGlnGluIleGlnGlnAspAsp                             151015                                                                        AspThr                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:50 base pairs                                                      (B) TYPE:nucleic acid                                                          (C) STRANDEDNESS:single                                                      (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:Other nucleic acid Synthetic DNA                           (iii) HYPOTHETICAL:YES                                                        (iv) ANTI-SENSE:YES                                                           (v) FRAGMENT TYPE:N-terminal fragment                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GTATCATCATCCTGCTGGATTTCCTGGGCGGTGCGTGGCTTGCCAACGTT50                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:2135 base pairs                                                   (B) TYPE:nucleic acid                                                         (C) STRANDEDNESS:double                                                       (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:Genomic DNA                                                (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:Corynebacterium glutamicum                                       (B) STRAIN:ATCC13032                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:mat peptide                                                      (B) LOCATION:514 to 1806                                                      (C) IDENTIFICATION METHOD:E                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GTTAACGGTTGTGAAAACTCTTTTAAGAAAAGCACTCTGACTACCTCTGGAATCTAGGTG60                CCACTCTTCTTTCGATTTCAACCCTTATCGTGTTTGGCGATGTGATCAGACTAAGTGATC120               ACCGTCACCAGCAAAAGGGGTTTGCGAACTTTACTAAGT CATTACCCCCGCCTAACCCCG180              ACTTTTATCTAGGTCACACCTTCGAAACCTACGGAACGTTGCGGTGCCTGCATTTTCCCA240               TTTCAGAGCATTTGCCCAGTACATCCGTACTAGCAACTCCCCCGCCCACTTTTTCTGCGA300               AGCCAGAACTTTGCAA ACTTCACAACAGGGGTGACCACCCGCACAAAACTTAAAAACCCA360              AACCGATTGACGCACCAATGCCCGATGGAGCAATGTGTGAACCACGCCACCACGCAAACC420               GATGCACATTACGTCGAAACAGTGACAGTGCATTAGCTCATACTTTGTGGTGGCACCGCC 480              CATTGCGAATCAGCACTTAAGGAAGTGACTTTGATGTCAAACGTTGGAAAGCCA534                     MetSerAsnValGlyLysPro                                                         1 5                                                                           CGTACCGCACAGGAAATCCAGCAGGATTGGGACACCAACCCTCGTTGG582                           ArgThrAlaGlnGluIleGlnGlnAspTrpAspThrAsnProArgTrp                              101520                                                                         AACGGCATCACCCGCGACTACACCGCAGACCAGGTAGCTGATCTGCAG630                          AsnGlyIleThrArgAspTyrThrAlaAspGlnValAlaAspLeuGln                              253035                                                                        GGTTCCGT CATCGAGGAGCACACTCTTGCTGCCGCGGCTCAGAGATCC678                          GlySerValIleGluGluHisThrLeuAlaAlaAlaAlaGlnArgSer                              40455055                                                                      TCTG GGACGCAGTCACCCAGGAAGGTGACGGATACATCAACGCTTGGC726                          SerGlyThrGlnSerProArgLysValThrAspThrSerThrLeuGly                              606570                                                                        GCA CTCACCGGTAACCAGGCTGTTCAGCAGGTTCGTGCAGGCCTGAAG774                          AlaLeuThrGlyAsnGlnAlaValGlnGlnValArgAlaGlyLeuLys                              758085                                                                        GCTGTC TACCTGTCCGGTTGGCAGGTCGCAGGTGACGCCAACCTCTCC822                          AlaValTyrLeuSerGlyTrpGlnValAlaGlyAspAlaAsnLeuSer                              9095100                                                                       GGCCACACCTA CCCTGACCAGTCCCTCTACCCAGCGAACTCCGTTCCA870                          GlyHisThrTyrProAspGlnSerLeuTyrProAlaAsnSerValPro                              105110115                                                                     AGCGTCGTTCGTCGCATCA ACAACGCACTGCTGCGTTCCGATGAAATC918                          SerValValArgArgIleAsnAsnAlaLeuLeuArgSerAspGluIle                              120125130135                                                                  GCACGCACCGAAGCG ACACCTCCGTTGACAACTGGGTTGTCCCAATCG966                          AlaArgThrGluAlaThrProProLeuThrThrGlyLeuSerGlnSer                              140145150                                                                     TCGCGGACGGCGAAG TGGCTTCGGTGGAGCACTCAACGTCTACAACTC1014                         SerArgThrAlaLysTrpLeuArgTrpSerThrGlnArgLeuGlnLeu                              155160165                                                                     CAGAAGGCAATGATCGC AGCTGGCGCTGCAGGCACCCACTGGGAAGAC1062                         GlnLysAlaMetIleAlaAlaGlyAlaAlaGlyThrHisTrpGluAsp                              170175180                                                                     CACGTCGCTTCTGAAAAGAAGT GTGGCCACCTCGGCGGCAAGGTTCTG1110                         HisValAlaSerGluLysLysCysGlyHisLeuGlyGlyLysValLeu                              185190195                                                                     ATCCCAACCCAGCAGCACATCCGCACCCTG AACTCTGCCCGCCTTGCA1158                         IleProThrGlnGlnHisIleArgThrLeuAsnSerAlaArgLeuAla                              200205210215                                                                  GCAGACGTTGCAAACACCCCAACTGTT GTTATCGCACGTACCGACGCT1206                         AlaAspValAlaAsnThrProThrValValIleAlaArgThrAspAla                              220225230                                                                     GAGGCAGCAACCCTGATCACCTCTGA CGTTGATGAGCGCGACCAACCA1254                         GluAlaAlaThrLeuIleThrSerAspValAspGluArgAspGlnPro                              235240245                                                                     TTCATCACCGGTGAGCGCACCGCAGAAG GCTACTACCACGTCAAGAAT1302                         PheIleThrGlyGluArgThrAlaGluGlyTyrTyrHisValLysAsn                              250255260                                                                     GGTCTCGAGCCATGTATCGCACGTGCAAAGTCC TACGCACCATACGCA1350                         GlyLeuGluProCysIleAlaArgAlaLysSerTyrAlaProTyrAla                              265270275                                                                     GATATGATCTGGATGGAGACCGGCACCCCTGACCTGGAGCTC GCTAAG1398                         AspMetIleTrpMetGluThrGlyThrProAspLeuGluLeuAlaLys                              280285290295                                                                  AAGTTCGCTGAAGGCGTTCGCTCTGAGTTCCCAGACCA GCTGCTGTCC1446                         LysPheAlaGluGlyValArgSerGluPheProAspGlnLeuLeuSer                              300305310                                                                     TACAACTGCTCCCCATCCTTCAACTGGTCTGCACACC TCGAGGCAGAT1494                         TyrAsnCysSerProSerPheAsnTrpSerAlaHisLeuGluAlaAsp                              315320325                                                                     GAGATCGCTAAGTTCCAGAAGGAACTCGGCGCAATGGGC TTCAAGTTC1542                         GluIleAlaLysPheGlnLysGluLeuGlyAlaMetGlyPheLysPhe                              330335340                                                                     CAGTTCATCACCCTCGCAGGCTTCCACTCCCTCAACTACGGCATG TTC1590                         GlnPheIleThrLeuAlaGlyPheHisSerLeuAsnTyrGlyMetPhe                              345350355                                                                     GACCTGGCTTACGGATACGCTCGCGAAGGCATGACCTCCTTCGTTGAC16 38                         AspLeuAlaTyrGlyTyrAlaArgGluGlyMetThrSerPheValAsp                              360365370375                                                                  CTGCAGAACCGTGAGTTCAAGGCAGCTGAAGAGCGTGGCTTCACCGCT 1686                         LeuGlnAsnArgGluPheLysAlaAlaGluGluArgGlyPheThrAla                              380385390                                                                     GTTAAGCACCAGCGTGAGGTTGGCGCAGGCTACTTCGACCAGATCGCA 1734                         ValLysHisGlnArgGluValGlyAlaGlyTyrPheAspGlnIleAla                              395400405                                                                     ACCACCGTTGACCCGAACTCTTCTACCACCGCTTTGAAGGGTTCCACT 1782                         ThrThrValAspProAsnSerSerThrThrAlaLeuLysGlySerThr                              410415420                                                                     GAAGAAGGCCAGTTCCACAACTAGGACCTACAGGTTCTGACAATTTAAATCTCC1836                     GluGluGlyGlnPheHisAsnXaa                                                     425430                                                                        CTACATCTGTACAACGGATGTAGGGAGTTTTTCCTTATATATGCCCTCCACAAATCCCCT1896              ATCGTGTGAGATGTGTTTCATAGGTGCCCCCAACGTTGCCTGTTGACTGCAAATTTT CCG1956             AAAGAATCCATAAACTACTTCTTTAAGTCGCCAGATTAAAGTCGTCAATGAAAGGACATA2016              CATGTCTATTTCCCGCACCGTCTTCGGCATCGCAGCCACCGCAGCCCTGTCTGCAGCTCT2076              CGTTGCGTGTTCTCCACCTCACCAGCAGGATTCC CCAGTCCAGCGCACCAATGAGATCT2135              (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:702 base pairs                                                     (B) TYPE:nucleic acid                                                         (C) STRANDEDNESS:double                                                       (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:Genomic DNA                                                (v) FRAGMENT TYPE:N-terminal fragment                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:Corynebacterium glutamicum                                       (B) STRAIN:ATCC13032                                                          (ix) FEATURE:                                                                 (A) NAME/KEY:transit peptide                                                  (B) LOCATION:514 to 702                                                       (C) IDENTIFICATION METHOD:E                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GTTAACGGTTGTGAAAACTCTTTTAAGAAAAGCACTCTGACTACCTCTGGAATCTAGGTG60                CCACTCTTCTTTCGATTTCAACCCTTATCGTGTTTGGC GATGTGATCAGACTAAGTGATC120              ACCGTCACCAGCAAAAGGGGTTTGCGAACTTTACTAAGTCATTACCCCCGCCTAACCCCG180               ACTTTTATCTAGGTCACACCTTCGAAACCTACGGAACGTTGCGGTGCCTGCATTTTCCCA240               TTTCAGAGCATTTGC CCAGTACATCCGTACTAGCAACTCCCCCGCCCACTTTTTCTGCGA300              AGCCAGAACTTTGCAAACTTCACAACAGGGGTGACCACCCGCACAAAACTTAAAAACCCA360               AACCGATTGACGCACCAATGCCCGATGGAGCAATGTGTGAACCACGCCACCACGCAAACC 420              GATGCACATTACGTCGAAACAGTGACAGTGCATTAGCTCATACTTTGTGGTGGCACCGCC480               CATTGCGAATCAGCACTTAAGGAAGTGACTTTGATGTCAAACGTTGGAAAGCCA534                     Met SerAsnValGlyLysPro                                                        15                                                                            CGTACCGCACAGGAAATCCAGCAGGATTGGGACACCAACCCTCGTTGG582                           ArgThrAlaGlnGluIleGlnGlnAspTrpAs pThrAsnProArgTrp                             101520                                                                        AACGGCATCACCCGCGACTACACCGCAGACCAGGTAGCTGATCTGCAG630                           AsnGlyIleThrArgAspTyrThrAlaAspGlnValA laAspLeuGln                             253035                                                                        GGTTCCGTCATCGAGGAGCACACTCTTGCTGCCGCGGCTCAGAGATCC678                           GlySerValIleGluGluHisThrLeuAlaAlaAlaAlaGlnArg Ser                             40455055                                                                      TCTGGGACGCAGTCACCCAGGAAG702                                                   SerGlyThrGlnSerProArgLys                                                      60                                                                        

What is claimed is:
 1. An isolated DNA which consists of a DNA from the isocitrate lyase gene of a coryneform bacterium, said DNA having at least a functional nucleotide sequence of nucleotide sequence 1 to 702 of SEQ ID NO:4, which regulates expression of a structural gene encoding a protein by repressing expression when carbon sources in a culture medium are sugars and inducing expression when carbon sources in a culture medium are non-sugars or a medium contains no sugar when functionally incorporated into a vector DNA together with said structural gene and introduced into a host coryneform bacterium.
 2. The DNA according to claim 1, wherein said structural gene is a gene encoding an enzyme or a protein selected from the group consisting of isocitrate lyase, β-galactosidase, chloramphenicol acetyltransferase, insulin, growth hormone, interferon and granulocyte colony stimulating hormone.
 3. The DNA according to claim 1, wherein said isocitrate lyase gene is obtained from a coryneform bacterium belonging to the genus Corynebacterium, Brevibacterium or Microbacterium.
 4. The DNA according to claim 3, wherein said coryneform bacterium is selected from the group consisting of Corynebacterium glutamicum ATCC 13032, Corynebacterium acetoacidophilum ATCC 13870, Corynebacterium acetoglutamicum ATCC 15806, Corynebacterium callunae ATCC 15991, Corynebacterium herculis ATCC 13868, Corynebacterium melassecola ATCC 17965, Corynebacterium lilium ATCC 15990, Brevibacterium immariophilum ATCC 14068, Brevibacterium saccharolyticum ATCC 14066, Brevibacterium thiogenitalis ATCC 19240, Brevibacterium divaricatum ATCC 14020, Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum ATCC 13869, Brevibacterium roseum ATCC 13825 and Microbacterium ammoniaphilum ATCC
 15354. 5. The DNA according to claim 1, wherein said host coryneform bacterium belongs to the genus Corynebacterium, Brevibacterium or Microbacterium.
 6. The DNA according to claim 5, wherein said host coryneform bacterium is selected from the group consisting of Corynebacterium glutamicum ATCC 13032, Corynebacterium acetoacidophilum ATCC 13870, Corynebacterium acetoglutamicum ATCC 15806, Corynebacterium callunae ATCC 15991, Corynebacterium herculis ATCC 13868, Corynebacterium melassecola ATCC 17965, Corynebacterium lilium ATCC 15990, Brevibacterium immariophilum ATCC 14068, Brevibacterium saccharolyticum ATCC 14066, Brevibacterium thiogenitalis ATCC 19240, Brevibacterium divaricatum ATCC 14020, Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum ATCC 13869, Brevibacterium roseum ATCC 13825, Brevibacterium ammoniagenes ATCC 6872 and Microbacterium ammoniaphilum ATCC
 15354. 7. A recombinant DNA prepared by incorporating into a vector DNA (1) a DNA which consists of a DNA from the isocitrate lyase gene of a coryneform bacterium said DNA having at least a functional nucleotide sequence of nucleotide sequence 1 to 702 of SEQ ID NO: 4, which regulates expression of a structural gene encoding a protein by repressing expression when carbon sources in a culture medium are sugars and inducing expression when carbon sources in a culture medium are non-sugars or a medium contains no sugar when incorporated into said vector DNA together with said structural gene and introduced into a host coryneform bacterium and (2) a structural gene encoding a protein.
 8. A coryneform bacteria transformant host carrying a recombinant DNA prepared by incorporating into a vector DNA autonomously replicable in said host (1) a DNA sequence from the isocitrate lyase gene of a coryneform bacterium, said DNA sequence having at least a functional nucleotide sequence of nucleotide sequence 1 to 702 of SEQ ID NO: 4, which regulates expression of a structural gene encoding a protein by repressing expression when carbon sources in a culture medium are sugars and inducing expression when carbon sources in a culture medium are non-sugars or a medium contains no sugar when incorporated into said vector DNA together with said structural gene and introduced into said host coryneform bacterium and (2) a structural gene encoding a protein. 